1. Field of the Invention
The present invention relates to a stage for holding porous media (filters) or non-porous media, that are flexible or rigid, and that carry multiple samples which may be biological or otherwise, and also to a method of analyzing said samples using the above-mentioned stage. The stage is suitable for placing beneath an optical microscope and may constitute an over-stage, or else it may replace the conventional stage. The stage of the invention is particularly suitable for analyzing samples rapidly, in particular by making use of automatic image analysis by computer.
2. Description of the Prior Art
It is recalled that in microscopy the object rests on a glass slide and, in general, it is covered by a glass cover-slip. When analyzing bacteria on a filter, this additional medium is naturally present, given that the only possible technique presently known for ensuring that the filter is properly plane is to mount it between a slide and a cover-slip.
Conventionally, a microscope stage includes a fixing plane which is fixed to a focusing rack. The plane of the stage moves in the Y direction and the slide is held by a vice that is movable in the X direction. The slide is placed against the stage and a spring Jaw is pressed laterally against the slide. In practice, this system suffers from two defects:
1) the glass slide may be at a slight angle relative to the plane of the stage; and
2) the lateral stress exerted by the spring may curve the slide slightly.
At high magnification, and when performing X and Y displacement, it thus becomes essential to keep on readjusting the focus, to such an extent that a microscope operator generally works with one hand constantly active on the micrometer screw.
The ever increasing use of image-analyzing computers coupled to microscopes has led to X-Y stages being used that are driven by stepper motors. On the more sophisticated models, a special over-stage stresses the slide better.
Regardless of whether conventional analysis or automatic image analysis is being performed, the system includes a glass slide (with the commonest size being 75 mm.times.25 mm.times.1.2 mm) that will never be accurately plane given that the slide is very thin (1.2 mm) and very long (75 mm). In addition, slides are cheap consumable objects and they are not required to have very special optical properties.
It should be observed that the thinness of the slide is desirable for optical reasons. In conventional microscope applications, light arrives from below (transmitted light) via a condenser. To ensure that the illumination is intense and uniform, it is necessary to bring the condenser as close to the object as the objective lens.
This is not possible at very high magnification. An objective lens having a magnification of 100 times (giving a total magnification of 2000) is disposed at a distance of about 0.2 mm from the object (which leaves only just enough room for the cover-slip over the object).
Naturally, when using epifluorescence, there is no need for a condenser since the illumination arrives via the objective lens.
With filters, mounting a filter between a slide and a cover-slip is the only available technique for making the filter properly plane.
Filtration is being used more and more for analyzing particles contained in gases and liquids. These particles are varied in nature, being biological, mineral, metallic, etc. . . . One example is separating and counting bacteria in milk (using filters known under trade names "NUCLEOPORE", "MULLIPORE", etc.).
It should also be recalled that a liquid mounting medium is required, constituting either by oil, or by a viscous medium (Canada balsam) or else by a medium that sets over time.
The corresponding operations are relatively difficult and not very compatible with notions of high analysis throughput.
In addition, it is very difficult to obtain an accurately plane preparation. The filter cannot be pressed down accurately and the inevitable differences in the thickness of the above-mentioned mounting liquid cause the cover-slip itself to be deformed (which is not very serious if immersion oil is interposed between the objective lens and the cover-slip).
It should therefore be observed that it is theoretically not possible merely to place the filter on a conventional glass slide for direct observation while dry. The undulations of the filter would be too big. However, there would be immense advantages in terms of simplicity and said if such observation were possible.
Thus, it may be observed that automatic image analysis which gives the best performance finds its performance degraded by the unsuitability of the methods of preparing and observing the sample. J-C. BISCONTE has described elsewhere a method making use of transparent films for continuous observation by image analysis (cf. French patent FR-2 565 350 and the patent issued on Euro-PCT patent application WO 85/05563).
For each slide, it is necessary to initialize image analysis, in particular to verify focusing.
This focusing problem is the most difficult part of analyzing small particles, and in particular for analyzing bacteria.
In this context, it may be recalled that bacteria may sometimes measure less than one micron, and that under the best observation conditions, the depth of field is about half a micron. Thus, a defect in planeness of about one micron suffices to take bacteria completely out of focus, and therefore make them unsuitable for automatic analysis.
It is possible in image analysis to perform an automatic search for the best focus, however this process takes time. It requires 3 to 10 positions of stage height to be tested (in increments of 0.25 microns) in order to find the best adjustment (greatest degree of sharpness in the observed objects).
If the surface of the filter is subject to large fluctuations in planeness, e.g. of about 50 microns, then the image analysis faced with an empty field must be capable of deciding whether the field really is free from bacteria or whether the field is badly out of focus. It is quite normal to spend 10 to 20 seconds analyzing such a field! . . .
Unfortunately, such fields cannot be "skipped" since that runs the risk of obtaining completely erroneous results.
Patent application GB-2 141 254 (Miles) describes a device for holding an object to be observed stably in position on an observation window. To this end, that device comprises in combination:
a plate having mutually parallel main faces and an optical observation window of transparent material centrally disposed in a through opening therein; PA1 an annular suction gap surrounding the optical observation window; and PA1 a duct connecting the annular suction gap to a vacuum source (or source of reduced pressure). PA1 1) it makes it possible to obtain a medium for analysis that is accurately plane; PA1 2) it is particularly suitable for automatically and rapidly holding one or more samples in indexed manner for easy analysis (disposed on the same medium and always presented in the same X, Y, and Z manner) for the purpose of automatic image analysis by computer or for the purpose of manual analysis; PA1 3) it is also suitable for automatically searching for the best focus; PA1 4) it is compatible with observation both by transmission and by epifluorescence; PA1 5) it enables the medium to be observed directly without a cover-slip, thereby making it possible to use microscope objective lenses having very short focal lengths, thus obtaining better performance; PA1 6) it is compatible with dry observation and with observation under an immersion oil; and PA1 7) while being particularly adapted to optical microscopy, it may also be applied to electron microscopy or to other means of observation. PA1 an airtight annular gap provided at least around the plate and delimited between said plate, the stage, and at least an annular peripheral portion of the medium projecting beyond the plate; PA1 a device for applying suction to at least said annular peripheral portion of the medium to be analyzed, by creating a vacuum; and PA1 at least one airtight channel provided in the stage and putting the airtight suction gap into communication with the device for creating a vacuum; PA1 the plate is fixed to a radially inner end of an annular seat projecting from a portion of the wall delimiting the hole through the stage, the observation face of the plate against which the medium is pressed being at the same level as the top face of the stage when said plate is disposed in said hole, said plate having a radially outer surface portion spaced apart from the wall of the hole by said seat such that the airtight suction gap is delimited between: said radially outer surface portion of the plate, a surface portion of the seat on which the plate is fixed, said portion of the wall delimiting the hole through the stage, and said annular peripheral portion of the medium overlying in airtight manner the gap between the plate and the wall of the hole through the stage, the channel connecting the device for creating a vacuum to the airtight gap for applying suction to at least the annular peripheral portion of the medium being constituted by at least one blind groove formed in the stage and including a seat for receiving an airtight closure strip (for this suction groove) provided with a suction orifice for coupling to the above-mentioned device for creating a vacuum. PA1 an airtight annular gap provided at least around the plate and delimited between said plate, the stage, and at least an annular peripheral portion of the medium projecting beyond the plate; and PA1 a device for applying suction to at least said peripheral zone of the medium to be analyzed by establishing an electromagnetic field suitable for magnetizing at least said annular peripheral portion of the medium, which is fitted with or made of an annular band of ferromagnetic material. PA1 a device for applying suction to at least an annular peripheral zone of the medium to be analyzed by establishing a vacuum, which device is in communication with said peripheral zone of the medium, the observation plate being porous at least in a portion corresponding to said peripheral zone, said peripheral zone of the medium being pressed against the observation plate.
More precisely, Miles sets out essentially to replace the conventional slide carrier of a microscope which is unsuitable for supporting a flexible film, and also to avoid touching the top main face of the film so as to prevent any contamination or scratching thereof, and as a result Miles should be considered as constituting the technological background of the present invention while also illustrating the closest state of the art to the invention.
An object of the present invention is thus to provide a stage for holding porous media (filters) or non-porous media, that are flexible or rigid, and that carry samples which my be biological or otherwise, the stage satisfying practical requirements better than a conventional stage, and in particular:
A preferred application of the stage of the invention is constituted by counting bacteria on "NUCLEOPORE" type microporous filters.